It has become increasingly apparent that the transport of β-amyloid (Aβ) between the central nervous system (CNS) and plasma plays a key role in the regulation of brain β-amyloid levels, with Aβ being efficiently transported between the CNS and CSF (cerebral spinal fluid), CSF and blood and CNS and blood (Shibata (2000); Zlokovic (2004); Deane and Zlokovic (2007)). Therefore active vaccination with Aβ peptides or passive administration of specific Aβ antibodies rapidly binds peripheral Aβ altering the dynamic equilibrium between plasma, CSF and ultimately the CNS. This principle can also be applied to other areas where barriers potentially prevent the free exchange of Aβ or uptake of antibodies themselves such as the blood retinal barrier and access to the plaque-like deposits, or drusen, formed beneath the basement membrane of the retinal pigment epithelial (RPE) cell layer and the inner layer of the Bruch's membrane (Luibl et al (2006)). It has been postulated that through binding of β-amyloid in the periphery in blood, a so-called “sink” can be established by applying a physical gradient across the barrier leading to a lowering of β-amyloid levels within the compartment and its environment leading in turn to a reduction in Aβ-associated pathology (DeMattos et al (2002)). This could provide benefit in terms of cell survival by for example preventing cytotoxic effects of certain forms of β-amyloid. On the other hand antibodies have also been shown to penetrate across the blood brain barrier and hence may also elucidate effects by entering directly into the environment of the cells affected by β-amyloid related toxicity and act by removing or neutralising β-amyloid, by preventing deposition or by altering amyloid precursor processing (Deane et al. (2005); Bard et al (2000); Rakover et al (2006))
Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world. There are two major clinical presentations of AMD. Atrophic (dry) AMD is characterised by the degeneration of retinal pigment epithelial (RPE) and neuroretina. The early stages of atrophic AMD are associated with the formation of drusen, under the RPE cell layer. Early atrophic AMD can progress to an end stage disease where the RPE degenerates completely and forms sharply demarcated areas of RPE atrophy in the region of the macula: “geographic atrophy”. In this form of the disease, the degeneration of RPE results in the secondary death of macular rods and cones and in these cases this leads to the severe age-related vision loss. A proportion of AMD patients develop what can either be regarded as a different form or a further complication of the disease. Approximately 10-20% of AMD patients develop choroidal neovascularisation, (CNV). When this occurs the form of the disease is known as “wet AMD” and this can be associated with some of the most severe vision loss. In wet AMD, new choroidal vessels grow through breaks in Bruch's membrane and proliferate into and under the RPE and neuroretina. In typical cases, atrophic AMD develops in the eye before the development of the wet form, however, on infrequent occasions, the neovascular form can develop in the absence of prior development of the atrophic form. In both forms of the disease, vision loss occurs due to the death of photoreceptor cells, although in wet AMD internal bleeding from the leaky vessels formed during CNV also causes vision loss. In terms of therapy for AMD there has been some progress in developing novel treatments to address some aspects of wet AMD, in particular the reduction of leaky vessel bleeding from CNV by various molecules that inhibit either VEGF, (vascular endothelial growth factor) or the VEGF receptor signalling pathway. However, currently there are no definitive means of treatment for the very prevalent atrophic form of AMD nor to prevent the progression of early dry AMD either to geographic atrophy or to wet AMD, (Petrukhin K (2007)).
There are considerable similarities between the formation of drusen in AMD and in the formation of plaques in Alzheimer's disease (AD). Drusen have been found from histopathological and proteomic studies to contain similar types of protein components to AD plaques. The presence of apolipoprotein E and β-amyloid, (Aβ), protein as components of drusen in atrophic AMD suggests some sharing of the pathways of AD plaque and AMD drusen formations. The Aβ found in drusen is thought to be locally derived from the RPE cells. The involvement of the ageing process and also secondary inflammatory arrest also appears to be linked in both AMD and AD. In the inflammatory process associated with AMD there is an associated rise in expression of acute-proteins such as C-reactive protein (CRP) and Aβ protein. Both of these proteins are part of the pentraxin protein class and they may both induce complement activation and the activation of pro-inflammatory cytokines. Activated complement components are also found in drusen and a number of polymorphisms in genes involved in the alternative complement pathway have been shown to be associated with the development of AMD. Many polymorphisms have been described especially in the key regulator complement factor H, (CFH), but also in Factor B, C2 and C3. The implication of such polymorphisms is that a dysfunctionally activated or regulated alternative complement pathway is associated with AMD. The activated complement components lead to the formation of a final membrane attack complex which can lyse cells and lead to the release of cytokines such as VEGF. In diseases such as AD the deposition of plaques containing Aβ protein and neurofibrillary tangles are known to activate the complement pathway so it is possible that the presence of Aβ protein in drusen has a similar effect in AMD, (Rodrigues E (2007); Johnson L V (2001); Hageman G S et al., (2001)). A recent study investigated the prevalence of AMD amongst patients with AD in the USA and found that approximately double the number of expected cases of both late stage and early stage AMD were found in AD patients, (Baumritter et al., (2007)). Conversely an earlier, prospective population-based study had identified an increased risk of developing AD in individuals with advanced AMD, (Klaver C C et al., (1999)).
Initial studies to characterise the β-amyloid present in drusen appeared to highlight some differences from the β-amyloid present in AD plaques. Some drusen were found to stain weakly with Congo red, but no drusen β-amyloid seemed to show the green birefringence of β-amyloids stained with Congo red and observed under polarized light as with AD plaque β-amyloid. Confirmation of the β-amyloid present in drusen could however be demonstrated by both crystal violet and thioflavin T staining. The conclusion from this initial work was that the data was strongly suggestive that drusen exhibits some of the characteristics of β-amyloid and contains several β-amyloid-associated proteins but that they do not contain the fibrils characteristic of true β-amyloids. However, this study was unable to detect Aβ peptide, the principal component of cerebral β-amyloid in AD, nor amyloid precursor protein, (APP) in drusen, (Mullins R F et al. (2000)). This was in contrast to an early study that had reported the reaction of drusen with monoclonal antibodies directed against Aβ peptide, (Loeffler K et al., (1995)).
Further very detailed characterisation of the β-amyloid present in drusen and the association with ageing and AMD has been performed (Johnson L V et al., (2002), Anderson D H et al., (2004)). The β-amyloid is found to be associated with a sub-structural vesicular component of drusen and this co-localizes with activated complement components, (complement C3), into so-called “amyloid vesicles” which could also be primary sites of complement activation where the β-amyloid deposition could trigger local activation of the complement cascade. β-Amyloid could therefore be an important component of the local inflammatory events that lead to RPE atrophy, drusen biogenesis and the pathogenesis of AMD. The ultra-structural organization and the histochemical staining properties of the β-amyloid-containing drusen were studied in 152 donor human eyes. The β-amyloid is found in spherical elements organised as concentric ring-like structures and these are common sub-structural components of drusen. The sub-structures are composed of a central core with one or more concentric inner rings and most of the immunoreactivity to Aβ is associated with the outer layers of densely packed spherical sub-units, where punctuate regions iC3b can also be identified. The spherical structures are found in hard drusen and range from 2-10 um in diameter and can be seen in both macular and peripheral drusen in donor eyes both with and without clinical AMD. A morphometric analysis was performed on the drusen from the 152 donor samples of which 82 had clinical evidence of AMD. Drusen load varied with age in this study. The percentage of donors with little or no drusen dropped dramatically with age and there was an increase in the percentage of donors with moderate to heavy drusen loads with age, especially in those donors over 70 years of age. For the donors with light drusen loads, the percentage of donors with evidence of Aβ assemblies was almost 50%, whereas, for those donors with moderate to heavy drusen loads the percentage with Aβ assemblies approached 100%. Around half of those donors without clinical AMD had drusen which showed evidence of Aβ assemblies, but for those donors with clinically defined AMD, around 100% possessed some drusen with the Aβ assemblies. Some drusen can be densely packed with “amyloid vesicles” and this can account for a significant proportion of their total volume. Other smaller drusen may contain only a single large vesicle which again may account for a large proportion of drusen mass. Vesicles were sometimes found in the process of budding or fusing. Aβ immunoreactivity was also found in the cytoplasm of RPE cells (Anderson D H et al., (2004)). Some RPE cells that are either displaced by or flank drusen contained structures that appear similar to the β-amyloid vesicles in drusen. The staining pattern of the Aβ derived from RPE was thought to be from degenerate RPE cells that were transitioning to the formation of mature drusen. Longitudinally, oriented fibril arrays which are characteristic of aggregated β-amyloid fibrils in the brain of AD patients could not be identified in the drusen. The structures found in drusen seemed to represent a new type of macromolecular assembly of Aβ and activated complement components. The presence of Aβ was confirmed in drusen and the expression of the amyloid precursor protein, (APP), from which it is derived was highlighted in RPE cells using a number of antibody reagents with documented binding activity in AD plaques:                (i) Mouse anti Aβ monoclonal antibody 6E10 with epitope in amino acids 1-16, (Chemicon),        (ii) Mouse anti Aβ monoclonal antibody 4G8 with epitope in amino acids 17-24, (Signet Laboratories, Dedham, Mass., USA),        (iii) Mouse anti APP monoclonal antibody 22C11 with epitope outside of the β-amyloid peptide but within amino acids 66-81 of human APP N-terminus, (Chemicon),        (iv) Goat anti APP polyclonal antibody which was raised against a peptide of amino acids 44-63 of human APP, (Chemicon).        
Both of the monoclonal antibodies 6E10 and 4G8 were able to label the amyloid vesicles in drusen. The APP-specific monoclonal antibody, (22C11), showed only some particulate staining of RPE cytoplasm and not labelling of the amyloid vesicles. Although some of the 6E10 positive vesicles did also faintly stain with the APP polyclonal antibody it is likely that Aβ peptide and not APP is the positive constituent of these particles (Johnson L V et al., (2002)). Structures of a similar size and morphology to the drusen associated amyloid vesicles have been described as being reactive to the 6E10 antibody in the brains of transgenic mice expressing the human APP protein (Terai K et al., (2001)).
A study to look at the presence of Aβ in the drusen of AMD patients compared to the drusen in normal retinas found Aβ immunoreactivity in 4/9 eyes with AMD and in 0/9 normal eyes (Dentchev T, et al., (2003)). The 9 AMD retinas consisted of 3 early AMD patients, 3 with geographic atrophy and 3 with neovascular AMD and the positive samples were 2 of those with early AMD and 2 with geographic atrophy with the largest quantity of Aβ being seen at the edges of the atrophy in the eyes with geographic atrophy. It was suggested that either the Aβ containing vesicles contribute to the adjacent RPE cell death or that they result from ongoing photoreceptor or RPE dysfunction or death, but that Aβ could play a role in both the early and the later stages of AMD.
It has been suggested in some experiments that non-fibrillar Aβ intermediates and not the insoluble fibrils are responsible for the primary cytotoxic effects of Aβ, (reviewed in Anderson D H et al., (2004)). It is possible that Aβ in drusen could both contribute to the local inflammatory processes involved in AMD by triggering complement activation and by assembling into macromolecular aggregates containing cytotoxic Aβ peptide forms which could result in direct killing of RPE and/or retinal ganglion cells (Anderson D H at al., (2004)). In a further study oligomeric Aβ was detected in drusen and this did not appear to co-localize with the Aβ containing vesicles described above in drusen (Luibl et al (2006)). In this study, oligomeric Aβ reactivity was seen in all drusen but not in eyes without drusen.
In addition to the aforementioned ways in which Aβ seems to be involved in the biology of drusen and its potential roles in the aetiology of AMD, Aβ has also been reported to directly interact with VEGF and this may also play a role in the pathogenesis of both AD and AMD. VEGF has been shown to co-localize with Aβ in the plaques of brains of patients with AD. VEGF has been shown to bind very strongly to Aβ but the binding does not seem to impair VEGF cell binding or VEGF mitogenic activity at least in vitro. The role of such VEGF binding in AD is not clear (Yang S P et al., (2004)). However, VEGF plays a clear role in the pathogenesis of AMD and potential localisation of high local levels of VEGF associated with Aβ could be implicated in the generation of CNV. Activation of the alternative complement pathway and activated complement components are thought to trigger VEGF expression, but evidence has also been published that Aβ can also induce VEGF expression in human RPE cells in vitro (Yoshida T et al., (2005)). Additionally, mice disrupted for the nephrilysin gene, which encodes a peptidase that degrades Aβ, have increased deposition of Aβ under the RPE and also show increased RPE cell degeneration (Yoshida T et al., (2005)).
There are no clear animal models for the generation of all of the AMD pathology but there have been some interesting findings and parallels with the human disease shown in the ocular phenotypes of transgenic mice that carry modifications of the apolipoprotein E (apoE) gene. Association of lipid carrying apoE protein to the apoE receptor 2 has recently been shown to trigger the endocytosis of APP in neuroblastoma cells, leading to the production of Aβ (He X at al., (2007)). Transgenic mice which have had the murine ApoE gene inactivated but which instead express human Apo E variants: Apo E3 Leiden, (Kliffen M, et al., (2000)), and especially Apo E4, show, when on a high fat diet, ocular phenotypes ranging from basal laminar deposits under the RPE to drusen deposition and CNV (Malek G, et al., (2005)). The eyes of aged targeted replacement mice apoE mice expressing human Apo E4 when placed on a high fat diet developed changes which mimic the pathology of human AMD: diffuse sub-RPE deposits, drusen, thickening of Bruch's membrane, RPE atrophy, hypopigmentation and hyperpigmentation. In some cases mice develop marked CNV and there is loss of visual acuity as measured by electroretinogram (ERG). The model also demonstrates the presence of murine Aβ both associated with the CNV and with the drusen-like deposits and the presence of elevated levels of murine VEGF. The model has been used to test the hypothesis that the intravenous injection of a monoclonal antibody to β-amyloid can be used to reduce drusen load in a similar way to the reduction of Aβ containing plaques in the brains of AD models and preliminary evidence suggests that drusen load was reduced in these mice upon intravenous administration of an anti-Aβ monoclonal antibody (Bowes Rickman C (2007) & Ding, J D et al. (2008)).
In summary, this provides evidence that β-amyloid may be a key factor in AMD pathology and disease. Although the exact mechanisms that cause the production of Aβ in RPE and the exact mechanism or mechanisms by which Aβ acts to influence AMD are not completely understood, the evidence implies that clearing of Aβ by agents that bind and potentially neutralise or just remove Aβ may provide a possible route to clearing drusen in AMD, reducing complement activation in AMD, reducing RPE atrophy and potentially reducing the induction of VEGF expression in RPE and its localisation at high levels around drusen. Such therapy could therefore provide means of preventing, delaying, attenuating or reversing the loss of vision due to AMD and its progression to geographic atrophy and/or exudative AMD. This may result in decreased levels of Aβ containing drusen and/or local Aβ in the surrounding environment of the RPE and thereby interfere in both the early and later stages of AMD and treat the underlying cellular decline that causes the loss of vision.
“Glaucoma type diseases” is a nonspecific term used for a group of diseases that can lead to damage to the eye's optic nerve and result in blindness. It is a major cause of blindness in the world caused ultimately by increased intraocular pressure (IOP) and decreased visual acuity. The link between IOP and how this leads to apoptosis of the retinal ganglion cells (RGC) is not well understood. High IOP alone can induce apoptosis (Cordeiro et al (2004); Quigley et al (1995)) but in itself is not the only cause of cell death of the optic neurons. In addition it has been observed that the vision can continue to deteriorate even after the normalisation of the IOP following treatment with eye pressure lowering agents (Oliver et al (2002)).
Recently there have been reports linking the potentially cytotoxic effects of β-amyloid to apoptosis of RGCs in glaucoma (McKinnon et al (2002)). In animal models of glaucoma it has been demonstrated that caspase-3 protease is activated in RGCs which leads to abnormal processing of amyloid precursor protein (APP) by caspase-3 generating potentially toxic fragments of APP including β-amyloid (McKinnon et al (2002); Cheung et al (2004)). Amongst other cells, RGCs have been shown to express APP and this therefore appears a plausible source of β-amyloid. Both elevated levels of APP and elevated levels of β-amyloid have been implicated with activating caspase-3 although this has been observed primarily in vitro systems. It is unclear whether APP levels in the RGCs are also increased in glaucoma thus contributing to the generation of even more β-amyloid in a positive feed back mechanism. Even more recently, the involvement of β-amyloid with apoptosis of RGCs in a rat model of glaucoma has been suggested (Guo et al (2007)). Several agents targeting β-amyloid or β-amyloid production were tested and showed a reduction of retinal ganglion cell death in vivo with a possible mild enhancement effect when all three treatments were used together. The largest effect was seen by using an anti-β-amyloid antibody which almost matched the effects seen with all three agents together.
In summary this provides evidence that β-amyloid may be a key factor in the pathology of glaucoma-type diseases. Although the exact mechanisms that cause the production of β-amyloid in RGCs and the connection with IOP are not completely understood, the evidence implies that clearing of β-amyloid by agents that bind and potentially neutralise or just remove β-amyloid may provide a possible route to preventing RGC apoptosis in glaucoma and therefore provide means of delaying, attenuating or reversing the loss of vision in glaucoma. This may result in decreased levels of β-amyloid in the RGCs and surrounding environment and thereby address the underlying cellular decline that causes the loss of vision.
β-Amyloid may play a role in other ocular diseases and has been associated with the formation of supra-nuclear cataracts especially in those seen in AD patients and the components of the Aβ generation and processing pathway are present in the lens (Goldstein L E, et al., (2003); Li G, et al., (2003)). The therapeutic approaches described for intervention in AMD and glaucoma-type diseases may therefore be applicable to the prevention of Aβ dependent cataract formation.